Abstract
BackgroundNotch signalling is essential for the development and maintenance of the colonic epithelium. Its inhibition induces a differentiation phenotype in vivo and reduces adenomas in APCmin mice. Whether Notch signals are also required in colorectal cancer (CRC) has remained elusive. Therefore, 64 CRC cell lines were analysed for the occurrence of proteolytically processed, active Notch.Results63 CRC lines contained a fragment with approximately the size of the Notch1 intracellular domain (NICD), which is required for signalling. Subsequent analyses with an antibody that specifically recognises the free Val1744 residue generated by γ-secretase-mediated cleavage of Notch1 showed that a subset of CRC cells lacks this specific Val1744-NICD. Surprisingly, inhibition of Val1744-NICD signalling with different γ-secretase inhibitors (GSI) did not lead to substantial effects on CRC cell line growth or survival. However, transient activation of Erk upon GSI treatment was detected. Since cisplatin relies on Erk activation for bioactivity in some cells, platinum compounds were tested together with GSI and enhanced cell killing in a subset of Val1744-NICD-positive CRC cell lines was detected. Erk inhibition ablated this combination effect.ConclusionWe conclude that γ-secretase inhibition results in activation of the MAP kinases Erk1/2 and, when used in conjunction, enhances cell death induced by platinum compounds in a large subset of colorectal cancer cell lines.Furthermore the activation of Erk appears to be of particular importance in mediating the enhanced effect seen, as its inhibition abrogates the observed phenomenon. These findings do not only highlight the importance of signalling pathway crosstalk but they may also suggest a new avenue of combination therapy for some colorectal cancers.
Highlights
Notch signalling is essential for the development and maintenance of the colonic epithelium
Size heterogeneity of Notch fragments in colorectal cancer (CRC) cells To gain insight into potential functions of Notch signalling in CRC cells, initially a panel of 64 CRC cell lines was analysed with an antibody raised against the C-terminus of Notch1 for the presence of a Notch fragment corresponding in size to the Notch1 intracellular domain (NICD), which is generated by γ-secretase cleavage of Notch
The only exception found was the CRC line HDC-9, which was examined by subcellular fractionation, but no NICD was detected
Summary
Notch signalling is essential for the development and maintenance of the colonic epithelium. Whether Notch signals are required in colorectal cancer (CRC) has remained elusive. 64 CRC cell lines were analysed for the occurrence of proteolytically processed, active Notch. The Notch signalling pathway, already discovered in 1919 by Thomas H. For the activation of Notch signalling, a number of proteolytic processing events are required, most notably the final cleavage of Notch by a multi-protein complex termed γ-secretase. This releases a defined fragment (Val1744-NICD) of the membrane bound Notch protein (page number not for citation purposes). Anti-Bcl (B46620) was from Transduction Laboratories (Lexington, KY, USA). Peroxidase-conjugated anti-mouse (715036-151) or anti-rabbit IgG (711-036-152) antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-Hes was a gift from Dr Tatsuo Sudo, Toray Industries, Kamakura, Japan
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